Construction Expression Vector Of The Fruit Ripening Correlation Gene LePelC And LeAPETALA2LIKE In Tomato And Analysis Of The Expression Pattern Of The Genes

Tomato(Lycopersicume sculuentum Mill) is one of the most important crops of the world and plays an important role in research because it is the model plant in transgene research.Tomato fruit is climacteric fruit.Climacteric ripening is characterized by an upsurge in the respiration rate accompanying an autocatalytic ethylene production peak during fruit ripening.The main life process of fruit after harvest is respiration.The characteristic and level of respiration affect the shelf life and disease resistance of fruits directly,and it can partially explain the deterioration and decay of fruits.Improvement of fruit storability is a key object in tomato breeding. During recent years,based on the knowledge of the molecular mechanism of tomato fruit ripening,prominence progress on fruit storability has been made by some ripening related mutant gene research and gene engineering method.Most of these research concentrate on 1-amino-cyclopropane-1-carboxylic acid Oxidase(ACO), 1-amino-cyclopropane-1-carboxylic acid Synthase(ACS) and R/N,NOR,GR genes, but relative little work has been done about the enzymes which can affect cell wall degradation and fruit soften directly.Zhangjun Fei(2004) had created a large tomato EST collection which contains＞150 000 individual EST sequences representing about 30000 unique genes from 27 different tissues/treatments.Large amounts of gene expression data are being generated using the public array based on the work of a public cDNA microarray that based on this EST collection.Fei had told us some candidate gene which may play important role during tomato fruit ripening.So, basing on the sequence information in public,we design experiments to clone two of the candidates and study the expression.pattern of these two genes in tomato fruit ripening by sime-quantitative PCR and Real-Time fluorescence PCR.The main results and conclusion are as follow:1 Cloning the two genes by RT-PCR methodBased on the gene sequence on GenBank we design specific prime by "primer 5.0" software to clone the two genes from eDNA that reverse transcripted from RNA. Both of the genes are 1.2kb.Baleta shows the EST TC 115905 is highly homology and the analysis of its amino acid sequence and Phylogenetic tree indicate it belong to pectate lyase super family,so we named it LePelC.Another EST TC124194 code an amino acid sequence contain AP2 conserve structure and is 57%homology to Petunia hyhrida PHAP2A protein,so we named it LeAPETALA2LIKE. 2 Study the expression pattern of LePelC and LeAPETALA2LIKE in tomato fruit ripening process by semi-quantitative PCR and quantitative PCRThe expression of tomato LePelC and LeAPETALA2LIKE genes on 17 ripening stage of fruit was studied by semi-quantitative PCR and quantitative PCR method. The results indicate LePelC has a basic level expression on the primary ripening stage and the level increasing rapidly on the 25^th day after pollination.And on the 28^th day after pollination the expression of LePelC reached a high level and the level was decrease as fruit ripe,but the level is still much higher than the primary ripening stage. Quantitative PCR further reveal the expression pattern of LePelC and all of the results support the product of this gene is tomato pectate lyase.On the other hand,the expression of LeAPETALA2LIKE is similar with LePelC.The results indicated the expression of this gene also had a surging with the appearance of respiration climacteric and it also had a basic expression on the primary ripening stage,but the level was much lower than LePelC.This result suggests LeAPETALA2LIKE is not an important gene during fruit ripping process.The study revealed the expression pattern of these two genes and we constructed the over-expression vector and ani-sense expression vector,of these two genes to study the function of the genes.We had transfered the expression vectors into Agrobacterium tumefaciens EHA 105 for the facility of further study.